1. Technical Field
The present invention relates to a process for preparing a retrovirus vector having a high titer and employed in gene therapy.
2. Prior Art
Owing to the remarkable progress in genetic engineering in recent years, there have been identified genes causative of a number of genetic diseases and thus the pathological mechanisms of these diseases have been clarified at the molecular level. Under these circumstances, studies on gene therapy have been made to transfer genes seemingly capable of ameliorating diseases into cells and some of these treatments have been already put into practical use. Also, attempts have been made to apply the gene therapy to the treatment of cancer, AIDS, etc. In gene therapy, there are known several methods for transferring foreign genes. Among all, the most frequently employed method at the present stage is the one with the use of retrovirus vectors (Miller, A. D., Blood, 76, 271–278, 1990). Use of these vectors has the following advantages. Since the transferred gene can be surely integrated into chromosomes, it can be expressed stably over a long period of time. In addition, this method is a highly safe one with little fear of cytotoxicity. On the other hand, this method suffers from some problems such that no gene can be transferred into cells in a number of cases because of the absence of any receptor for virus envelope proteins in the target cells, that large-sized DNAs cannot be inserted thereby, and that the gene transfer thereby is available exclusively into cells which are capable of dividing. Although gene therapy with the use of retroviruses has been frequently employed, no sufficient therapeutic effect can be achieved thereby hitherto because of the above problems (Marshall, E., Science, 269, 1050–1055, 1995).
To effect the gene therapy, anyway, it is required to satisfy at least the following three conditions: 1) to efficiently transfer a desired gene into the target cells; 2) to ensure the continuous expression of the transferred gene; and 3) to be safe for the environment including the patient.
The conventional process for preparing a retrovirus vector comprises transferring a retrovirus genome containing a desired foreign gene into cells called packaging cells wherein retrovirus gag, pol and env have been expressed stably to thereby give a retrovirus containing the foreign gene in its vector DNA. However, it is difficult to prepare vectors with such high qualities as usable for clinical purposes by this process. Thus, a number of studies have been made to prevent the occurrence of a replication competent retrovirus (RCR), to produce a retrovirus having a high titer, and to elevate the titer of a retrovirus vector by improving the vector genome structure or examining the conditions for condensation or gene transfer (Vile, R. G., Gene Therapy, Churchill Livingstone, 12–30, 1995). In spite of these efforts, no technique has been established so far for a vector having a broad infection range and a high titer in a large scale stably, which is one of serious obstacles to the gene therapy.
Meanwhile, studies have been energetically made for a long time by using vesicular stomatitis virus (VSV) as a model of pseudotyped viruses as the joint between retroviruses and other viruses (Zavada, J., Arch. Virol., 50, 1, 1976). The term “pseudotype” means a phenomenon wherein a virus genome germinates while being surrounded by the coat protein of another virus. VSV is a virus having a negative single-stranded RNA genome and belonging to the family Rhabdovirus. It is considered that the receptors on the cell surface of the coat protein (G protein) thereof include anionic lipids such as phosphatidylserine. Thus, it is known that VSV has an extremely broad host range. It is therefore assumed that by preparing a pseudotyped retrovirus having this VSV-G gene product in the coat, genes can be efficiently transferred into cells which can be transduced at only a low or even no infective efficiency with retroviruses having the inherent envelope protein. In fact, Emi et al. (Emi, N., et al., J. Virol., 65, 1202–1207, 1991) and Yee et al. (Yee, J. K., et al., Proc. Natl. Acad. Sci. USA, 91, 9564–9568, 1994) reported a process for preparing a retrovirus vector having a VSV-G gene product as its envelope and pointed out that this pseudotyped virus enabled efficient gene transfer into cells which could have been transduced only at a low infective efficiency with a retrovirus having the inherent envelope protein.
To clinically apply such a VSV-G pseudotyped virus vector, it is necessary to establish a method for acquiring a virus with a high titer at a high reproducibility. However, it is difficult to produce the VSV-G gene product at a high level and at a high reproducibility in packaging cells, since the VSV-G gene product per se has a cytotoxicity. This is a serious problem in the development of pseudotyped vectors which are expected to be widely applicable. Recently, it was reported that VSV-G pseudotyped virus vector-producing cells can be prepared by regulating the expression of the VSV-G gene product with the use of tetracycline (Yang, Y., et al. Hum. Gene. Ther., 6, 1203–1213, 1995; Chen, S. T., et al., Prc. Natl. Acad. Sci. USA, 93, 10057–10062, 1996; and Ory, D. S., et al., Proc. Natl. Acad. Sci. USA, 93, 11400–11406, 1996). However, there still remain some problems in these reports such that the regulation of the expression of the VSV-G gene product was not completely regulated by tetracycline and, therefore, the producing cells might be still re-infected with about 102 to 104 i.u./ml of the VSV-G pseudotyped virus vector thus produced; and that the stability of the packaging cells over a long period of time was still unreliable, since they were made of the co-transfection of a DNA with the VSV-G expression and another DNA with the drug-resistance gene expression.
It is also known that, when a foreign gene to be transferred into target cells with the use of a retrovirus strongly affects the cells, the virus carrying this foreign gene in its virus vector DNA cannot be recovered stably, since the foreign gene product affects in the virus-producing cells (i.e., the packaging cells containing and expressing the vector DNA) per se (Pear, W. S. et al., Proc. Natl. Acad. Sci. USA, 90, 8392–8396, 1993).
In the present description, the term “pseudotyped virus vector” refers to a retrovirus vector having a VSV-G gene product in its envelope, while the term “retrovirus vector” refers to both a retrovirus vector having the inherent envelope protein and a “pseudotyped virus vector”.
The term “prepackaging cells” refers to cells in which gag and pol of a retrovirus can be expressed and env thereof cannot be expressed in usual before recombinase was introduced to cells. The term “prepackaging cells containing a vector DNA” refers to the prepackaging cells as defined above into which a vector DNA has been transferred. Further, the term “packaging cells containing a vector DNA” refers to cells capable of producing a virus when a recombinase is transferred thereinto.
Moreover, the term “drug resistance gene” as used herein refers to all of low-efficient drug resistance genes, short-lived transcript drug resistance genes having a base sequence of a short-lived mRNA of a drug resistance gene and conventional drug resistance genes.